Pain is the cancer-related event that is most disruptive to a cancer patient's quality of life. Although bone cancer pain is one of the most severe and common sources of chronic cancer pain, relatively little is known about the mechanisms that generate and maintain this pain. The proposed experiments are designed to study the role of two chemokines, CCL2 and CCL3 in the genesis and maintenance of bone cancer pain using an established mouse bone tumor model and a novel ex vivo model of cancer pain. Four Specific Aims will be pursued: 1) Measure the spontaneous release of CCL2 and CCL3 in vivo and in vitro, from three different tumor types (osteosarcoma, fibrosarcoma and nonpainful melanoma). Based on preliminary data we hypothesize that CCL2 is released only by the fibrosarcoma and osteosarcoma, while CCL3 is released only by the osteosarcoma. 2) Identify the cell types that are producing CCL2 and CCL3 by using immunocytochemistry to localize each mediator to cells at the tumor site. We hypothesize that CCL2 will be localized to fibrosarcoma and osteosarcoma cells and to osteoclasts and macrophages at the tumor site, while CCL3 will be found predominately in the osteosarcoma cells. 3) Investigate the effect of CCL2 and CCL3 on the functional responses of DRG neurons using a novel in vitro tumor/bone-explant/DRG culture system. We hypothesize that CCL2 and CCL3 released from tumor cells or bone produce acute and long-term changes in stimulus- evoked Ca2+ transients that reflect changes in voltage dependent Ca2+ channels and TRPV1 receptors. We further hypothesize that the knockdown of these chemokines will prevent the effect of tumor cells on Ca2+ transients. 4) Determine whether manipulation of expression of CCL2 and CCL3 in tumors cells alters the pain producing phenotype of the cells. We hypothesize that knock-in of CCL2 and CCL3 in nonpainful melanoma cells will result in pain producing tumors, while knock-down of CCL2 and CCL3 in fibrosarcoma or osteosarcoma cells will reduce bone tumor-associated nociceptive responsiveness in behavioral pain tests. The proposed experiments are unique in that they will utilize the technique of microperfusion to directly measure algogens produced in vivo by different tumor types, while an innovative ex vivo tumor/bone-explant/DRG culture system will allow us to measure these substances in the culture media and to analyze their effects on DRG neurons. In addition the use of a new sleeping beauty transposon-shRNA procedure for long-term knockdown or production of these mediators will provide an excellent opportunity to investigate their role in nociception both in vitro and in vivo. Results will extend our knowledge of the mechanisms underlying tumor nociception and will provide a template for developing novel, life-enhancing treatments of cancer pain. For cancer patients, pain relief is central to a high quality of life while they battle the disease, yet current therapies are often distressingly ineffective for some types of cancer pain. To address this critical need and develop new therapies that successfully diminish cancer pain, we need to understand at the cellular level how cancer pain is generated and why it persists. This research focuses on two proteins, CCL2 and CCL3, that are secreted by tumors [and other tissues], and whose absence appears to be linked to decreased cancer pain. Use of an animal model of bone cancer pain and a new ex vivo cancer pain model to test the effects of CCL2 and CCL3 knockdown or antagonism will provide important data that may lead to the development of novel therapies for this dreaded condition.